A select group of lupus anti-DNA autoantibodies penetrate into living cells (1), and one unusual lupus autoantibody that penetrates cell nuclei without causing any apparent harm to normal cells or tissues, 3E10 (2), has been developed as a molecular delivery vehicle. Specifically, a 3E10 single chain variable fragment (scFv) with an enhancing mutation in CDR1 that increases DNA binding and efficiency of nuclear penetration has been used to carry cargo proteins including p53, Hsp70, and other antibody fragments into cell nuclei in vitro and in vivo (3-6). 3E10 scFv also has activity by itself and has been shown to inhibit DNA repair, sensitize cancer cells to DNA-damaging therapy, and to be toxic to BRCA2-deficient cancer cells (7). 3E10 scFv has potential to be used in molecular therapy approaches to diseases ranging from cancer to ischemic conditions such as stroke, and a greater understanding of the details of the mechanism by which it penetrates cell nuclei is important to further delineating the scope of its therapeutic applications.
Mutations in 3E10 that interfere with its ability to bind DNA also render the antibody incapable of nuclear penetration. In addition, 3E10 scFv has previously been shown capable of penetrating into cell nuclei in an ENT2-dependent manner, with efficiency of nuclear uptake greatly impaired in ENT2-deficient cells (8). Taken together, these findings suggest a link between cellular uptake of DNA and nuclear penetration by 3E10 scFv. Interestingly, when a 3E10 scFv-Hsp70 fusion protein (Fv-Hsp70) was administered intravenously to rats three hours after ligation of middle cerebral arteries to induce stroke, Fv-Hsp70 was found to selectively localize to regions of ischemic brain (9).
The invention involves the discovery of the mechanism by which some anti-DNA antibodies or fragments thereof penetrate the cell for use in better treating disease, disorders and conditions.